Isoenzymes are enzymatically active proteins catalyzing the same reaction and occurring in the same species, but differing in certain of their physical-chemical properties. For example, lactate dehydrogenase (L-lactate:NAD oxidoreductase, EC 1.1.1.27; LDH) is a tetrameric enzyme having a molecular weight in the range of 140,000 depending on the source. The enzyme is coded by two structural genes producing two subunits (H and M) which combine to form the active tetrameric LDH enzyme. The permutations of H and M subunits in the active enzyme lead to five isoenzymes, e.g., H.sub.4, H.sub.3 M, H.sub.2 M.sub.2, HM.sub.4 L and M.sub.4. Currently, nomenclature for the LDH isoenzymes is based on their relative migration in an electric field, i.e., electrophoresis. The order of increasing migration rate toward the anode is H.sub.4 &gt;H.sub.3 M&gt;H.sub.2 M.sub.2 &gt;HM.sub.3 &gt;M.sub.4 and they are designated as LD.sub.1, LD.sub.2, LD.sub.3, LD.sub.4 and LD.sub.5, respectively.
In recent years, a great deal of attention has focused on the diagnostic value of isoenzymes. Determinations of the serum levels of the isoenzymes of cholinesterase, amylase, creatine kinase, lactate dehydrogenase, alkaline phosphatase and hexosaminidase have been found to have clinical value. For example, the relative levels of these LDH isoenzymes in a patient's serum, in addition to the total LDH activity, is of diagnostic value. This importance is derived from the relative abundance of LD.sub.1 and LD.sub.2 isoenzymes in heart, kidney and erythrocytes, LD.sub.4 and LD.sub.5 in skeletal muscle and the liver, and LD.sub.3 in the spleen, lungs, pancrease, thyroid, adrenal gland and lymph nodes. Necrosis of cells in damaged organs results in the release of their respective enzymes into the blood stream. Thus, detection and quantitation of the LDH isoenzymes can give information pertinent to location of damaged tissue, e.g., myocardial infarction, liver disorders, etc. Saifer et al, Clinical Chemistry, Vol. 21, No. 3, pp. 334-42 (1975), describe a study of the relationship of various serum levels of hexosaminidase isoenzymes to carriers of Tay-Sachs disease. Also, the level of the cardiac-muscle isoenzyme of creatine kinase in serum is useful in diagnosing cardiac infarction (D. W. Mercer, Clinical Chemistry, Vol. 20, No. 1, pp. 36-40 (1974)).
In general, three approaches have been used in the determination of isoenzyme levels. They involve physical-chemical, electrophoretic and immunochemical methods. Since a large number of literature references describe research with LDH isoenzymes, the following discussion will focus on LDH although the approaches are relevant to other isoenzymes as well.
Wrobleroski et al, Annotations of the New York Academy of Science, Vol. 94, p. 912 (1961), describe the use of thermal denaturation as a method for determining LDH isoenzymes, making use of the differential stability to heat of the isoenzymes. Emerson et al, Journal of Clinical Pathology, Vol. 18, p. 803 (1965), describe the selective inhibition of LDH isoenzymes with reagents such as urea and oxalate. Warburton et al, Enzymologia, Vol. 26, p. 125 (1963), and Warburton et al, Nature, Vol. 198, p. 386 (1963), describe the use of organic solvents to precipitate LDH isoenzymes. U.S. Pat. Nos. 3,388,044 and 3,326,777 and Bishop et al, Proceedings of the National Academy of Science, Vol. 69, p. 1761 (1972), describe the use of pyruvate and lactate to differentially inhibit LDH isoenzymes. Cawley et al, American Journal of Clinical Pathology, Vol. 45, p. 737 (1966), describe electrophoretic techniques for separation of LDH isoenzymes. Nisselbaum et al, Journal of Biological Chemistry, Vol. 236, p. 401 (1961), describe a method for inhibiting LDH isoenzymes using antisera to human heart and liver enzymes.
The procedures described above generally suffer various deficiencies. The physical-chemical procedures suffer from lack of suffient specificity. For instance, they do not adequately distinguish between heart LDH isoenzymes, i.e., LD.sub.1 and LD.sub.2, and muscle LDH isoenzymes, i.e., LD.sub.4 and LD.sub.5. In addition, the procedures are time-consuming and tedious. For instance, in the relative heat-stability test, samples of serum to which NADH is added are heated to 57.degree. C. and 65.degree. C. for 30 minutes, after which the remaining enzyme activity is compared with that of an unheated sample. The control gives total LDH, while the difference between the activities of the control and the sample at 57.degree. C. gives a measure of the heat-labile enzyme, principally LD.sub.5. The activity of the heat-stable fraction, (LD.sub.1), is that of the sample heated at 65.degree. C., while the difference between the activities of the two heated samples is an index of the LD.sub.2, LD.sub.3 and LD.sub.4 isoenzymes. Although the information that one hopes to obtain is extremely useful, the temperature control, NADH concentration and protein concentration are parameters that affect the enzyme stability. Because these parameters are, in general, difficult to control, the results are not always reliable. However, this type of approach to isoenzyme assay is, in general, more rapid than electrophoretic procedures.
Electrophoretic methods suffer the disadvantage of being time-consuming and tedious. In addition to running the electrophoresis, the technician must also (1) prepare the reagents for the LDH reaction, and because such reagents are generally unstable, they must be prepared fresh for each run (2) incubate the electrophoresis plates after exposure to reagent solution, (3) apply a fixing wash to the samples and (4) make a densitometric reading. The entire operation may take a minimum of several hours and requires the attention of a technician. In addition, the staining procedure must be carefully controlled so as to prevent under- or overstaining which leads to erroneous results.
Immunochemistry has shown promise as a potential technique in the isoenzyme assay field. For instance, the use of LDH isoenzymes as antigens affords the production of antisera that are relatively specific discriminators among the isoenzymes. However, the production of active antibodies is elicited only when the native LDH enzyme is used as the antigen. If LD.sub.1 is the antigen, antisera for LD.sub.1 are produced with inhibitory effect on LD.sub.4 &lt;LD.sub.3 &lt;LD.sub.2. Conversely, if LD.sub.5 is used as the antigen, antisera specific for LD.sub.5 are produced with inhibitory effect on LD.sub.2 &lt;L.sub.3 &lt;L.sub.4. Moreover, there is evidence of nonneutralizing antibody formation which protects isoenzymes from antibody inactivation. Also, the production of antisera is time-consuming and its purification is difficult.
Thus, it can be appreciated that better ways for assaying isoenzymes are continuously being sought.